Detection and treatment of infectious and inflammatory lesions

ABSTRACT

A polyspecific anti-leukocyte antibody conjugate for targeting foci of leukocyte accretion comprises an immunoreactive polyspecific composite of at least two different substantially monospecific antibodies or antibody fragments, conjugated to at least one imaging agent, wherein at least two of said antibodies or antibody fragments specifically bind to different leukocyte cell types. 
     A method for targeting an imaging agent to an inflammatory or infectious lesion comprises injecting a mammal parenterally with an effective amount for targeting of the above anti-leukocyte imaging conjugate. 
     A therapeutic anti-leukocyte antibody-agent conjugate for targeting foci of leukocyte accretion comprises at least one immunoreactive substantially monospecific antibody or antibody fragment which specifically binds to at least one leukocyte cell type, conjugated to at least one therapeutic antimicrobial agent. 
     A method of treatment of an infectious lesion comprises injecting a mammal parenterally with an effective amount for therapy of the above anti-leukocyte therapeutic conjugate.

BACKGROUND OF THE INVENTION

This invention relates to reagents and methods for targeting at leastone diagnostic or therapeutic agent to an inflammatory or infectiouslesion. Polyspecific antibody composites specific for at least twodifferent leukocyte cell types are conjugated to at least one diagnosticor therapeutic agent for use in the present invention.

It has been recognized that, since leukocytes accumulate in largenumbers at localized sites of infection or inflammation, it might befeasible to detect such sites by removing leukocytes from the blood,labeling them with an appropriate indicator, conventionally In-111, andreturning them to the blood. After a period of time has passedsufficient to allow the labeled leukocytes to redistribute in the body,the subject is scanned with suitable equipment to detect localization ofthe labeled leukocytes. While effective, the method described above issubstantially time consuming since time is required for the leukocyteseparation, labeling and, particularly redistribution in the body afterre-injection.

In U.S. Pat. No. 4,634,586 (Goodwin et al.), incorporated herein byreference in its entirety, leukocytes are radioimmunoimaged by injectingpatients with an immunoreactive nonleukocidal conjugate of ananti-leukocyte monospecific antibody and a gamma emitting radioactivemetal chelate, waiting for the conjugate to localize on the leukocytes,injecting a patient with an antibody to the conjugate to clear the bloodof background nonlocalized conjugate, and visualizing the leukocytes byscintillation scanning.

Leukocyte imaging has been severely limited in the prior art due to poortarget to background ratio. It has been shown that the localizationratio can be increased by using second antibody clearance. However, thetarget to background ratio remains a problem, because each targetingantibody only binds to a specific leukocyte cell type, either agranulocyte, a monocyte, a B-lymphocyte or a T-lymphocyte. Therefore,there will be many antibodies that are highly reactive and specific fora particular leukocyte cell in the background that have not bound to thetarget site, because that particular leukocyte cell type is not presentin appreciable concentration at the site of infection or inflammation.

A need therefore continues to exist for a method of targeting an imagingor therapy agent to an inflammatory or infectious lesion with higherefficiency and enhanced target to background ratio to permit moreeffective detection and/or treatment of the lesion.

OBJECTS OF THE INVENTION

One object of the present invention is to provide a polyspecificantibody-agent conjugate which selectively binds to at least twodifferent leukocyte cell types, for targeting inflammations orinfectious lesions with an enhanced target to background ratio.

Another object of the present invention is to provide a method fortargeting a polyspecific antibody-agent conjugate to an infectious orinflammatory lesion with higher efficiency and an enhanced target tobackground ratio.

A further object of the invention is to provide reagents and methods formore efficient detection and/or therapy of infectious and inflammatorylesions.

Other objects of the present invention will become more apparent tothose of ordinary skill in the art in light of the following discussion.

SUMMARY OF THE INVENTION

These and other objects of the present invention are achieved byproviding a polyspecific antibody-agent conjugate for targeting foci ofleukocyte accretion, comprising an immunoreactive polyspecific compositeof at least two different substantially monospecific antibodies orantibody fragments, conjugated to at least one imaging agent, wherein atleast two of said antibodies or antibody fragments specifically bind todifferent leukocyte cell types.

The invention also provides a method for targeting a diagnostic agent toan inflammatory or infectious lesion which comprises injecting a mammalparenterally with an effective amount for targeting of the polyspecificantibody-agent conjugate.

The invention further provides an anti-leukocyte antibody-agentconjugate for targeting and treating an infectious lesion containing afocus of leukocyte accretion, comprising at least one immunoreactivesubstantially monospecific antibody or antibody fragment whichspecifically binds to at least one leukocyte cell type, conjugated to atleast one therapeutic antimicrobial agent.

A method for treating infectious lesions is also provided, comprisingparenterally injecting a therapeutically effective amount of theforegoing conjugate in a patient with such a lesion. Polyspecificanti-leukocyte antibody/fragment mixtures and/or composites also can beused as targeting vehicles for therapy according to the invention.

In addition, the present invention provides sterile injectablepreparations and kits for use in practicing the foregoing method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improvement over the prior art imagingmethod of Goodwin et al. through the use of a polyspecificantibody-agent conjugate for targeting foci of leukocyte accretion.Different leukocyte cell types (e.g., granulocytes, monocytes, T- andB-lymphocytes) that are involved in the development of an infectious orinflammatory lesion are often present in markedly different ratios inthe inflammatory or infectious lesion, depending upon the nature of theagent that initiates the development of the lesion and/or on the age ofthe lesion. Use of a monospecific antibody, as taught by Goodwin, willresult in inefficient targeting of the lesion if only a portion of theleukocyte population at the site of the lesion bind the targetingantibody, and this will reduce the target to background ratio (alsocalled "localization ratio"). Use of a mixture of antibodies withdifferent leukocyte specificities can improve the percentage of injecteddose reaching the target site if the right proportion of specificitiesis used, but can further increase binding to non-target leukocytes ifthe lesion contains primarily a single leukocyte cell type.

The present invention resolves this dilemma by using a polyspecifictargeting antibody composite which is able to bind to two or moredifferent leukocyte cell types. The imaging agent component of theantibody-agent conjugate is thereby localized at the target site withhigher efficiency and an enhanced target to background ratio, regardlessof the mix of leukocyte cell types.

The polyspecific targeting antibody composite comprises at least twodifferent substantially monospecific antibodies or antibody fragments,wherein at least two of the antibodies or antibody fragmentsspecifically bind to different leukocyte cell types. Thus, at least oneantigen binding site on the composite will bind to a first leukocytecell type while at least a second antigen binding site on the sametargeting composite will bind to a different leukocyte cell type. Such acomposite will be denoted an "anti-leukocyte composite " herein. Theantibody composite may also contain antibodies and/or fragments thatbind to two or more different antigens or epitopes of the same antigenon the same leukocyte cell type. The leukocyte cell types includegranulocytes, monocytes, B-lymphocytes and T-lymphocytes.

The immunological profile of the substantially monospecific, preferablymonoclonal, antibodies used to make the composite of the presentinvention can be adjusted to ensure optimal binding to infectious orinflammatory lesions and minimal binding to nontarget sites. Dependingupon the diagnostic use to which the reagent is to be put, the mix ofleukocyte cell type specificities, antigen specificities andspecificities for epitopes on antigens present on particular cell types,as well as of binding constants for the target antigens and/or celltypes, all can be used to fine tune the selectivity and targetingefficiency of the reagent according to the invention.

Imaging reagents according to the invention can comprise bispecific,trispecific or, more generally, polyspecific antibody/fragmentcomposites, conjugated to an imaging radioisotope or paramagneticspecies. The antibody component of the conjugate can be made with wholeantibodies or antibody fragments.

Use of term "antibody" herein will be understood to include antibodyfragments and thus to be equivalent to the term "antibody/fragment"which is used interchangeably therefor in this discussion. Antibodiescan be whole immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD, IgE,chimeric antibodies or hybrid antibodies with dual or multiple antigenor epitope specificities, or fragments, e.g., F(ab')₂, F(ab)₂, Fab', Faband the like, including hybrid fragments, and additionally includes anyimmunoglobulin or any natural, synthetic or genetically engineeredprotein that acts like an antibody by binding to a specific antigen toform a complex.

Antibodies can include antiserum preparations, preferably affinitypurified by conventional procedures, e.g., by binding antigen to achromatographic column pacing, e.g., Sephadex, passing the antiserumthrough the column, thereby retaining specific antibodies and separatingout other immunoglobulins and contaminants, and then recovering purifiedantibodies by elution with a chaotropic agent, optionally followed byfurther purification.

Monoclonal antibodies are also suitable for use in the presentinvention, and are preferred because of their high specificities. Theyare readily prepared by what are now generally considered conventionalprocedures for immunization of mammals with a immunogenic antigenpreparation, fusion of immune lymph or spleen cells with an immortalmyeloma cell line, and isolation of specific hybridoma clones. Moreunconventional methods of preparing monoclonal antibodies are notexcluded, such as interspecies fusions and genetic engineeringmanipulations of hypervariable regions, since it is primarily theantigen specificity of the antibodies that affects their utility in thepresent invention.

The present invention also envisions the use of antigen-specificfragments to create the polyspecific antibody-agent conjugate. Antibodyfragments can be made by pepsin or papain digestion of wholeimmunoglobulins by conventional methods. It is known that antibodyfragments may be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab')₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab' monovalent fragments. Alternatively anenzymatic cleavage using pepsin produces two monovalent Fab fragmentsand an Fc fragment directly. These methods are described, inter alia, byGoldenberg, in U.S. Pat. Nos. 4,036,945 and 4,331,647 and referencescontained therein, which patents are incorporated herein in theirentireties by reference, and in Nisonoff et al, Arch. Biochem. Biophys.,89, 230 (1960); Porter, Biochem. J., 73, 119 (1959); and Edelman et al,in "Methods in Immunology and Immunochemistry", Vol. 1, 422 (Acad.Press, 1967), and are conventional in the art.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments retain specificity to the leukocyteagainst which their parent antibodies are raised.

Antibodies to leukocyte antigens may be made by inoculating a host withleukocytes from the patient species. For instance, antibodies for use inhumans may be made by immunizing mice or other mammalian species withhuman leukocytes. Anti-human leukocyte serum may be collect from thehost and affinity purified to provide polyclonal antibody for making thecomposite. Alternatively, splenocytes may be taken from the immunizedhost and fused with a suitable tumor cell line using somatic cellhybridization techniques to produce hybridomas that produceanti-leukocyte antibodies. These hybridomas may be isolated, subclonedand cultivated to produce monoclonal antibodies.

Monoclonal antibodies or fragments that recognize and bind to aleukocyte antigen are available commercially or may be made from somaticcell hybridization techniques described originally by Kohler, B. andMilstein, C., Nature (1975) 256:495-497 and reviewed at length inMonoclonal Antibodies, Kennett, T. J., et al, eds, Plenum (1980).Commercially available monoclonal antibodies to leukocyte antigens arerepresented by: OKT anti-T monoclonal antibodies (available from OrthoPharmaceutical Company) which bind to normal T-lymphocytes; themonoclonal antibodies produced by the hybridomas having the ATCCaccession numbers HB44, HB55, HB12, HB78 and HB2; G7E11, W8E7, NKP15 andGO22 (Becton Dickinson); NEN9.4 (New England Nuclear); and FMC11 (SeraLabs).

The catalogue of Immunotech (Marseille, France, with worldwidedistribution including Pel Freeze, Brown Deer, WI, USA) listscommercially available monoclonal anti-leukocyte antibodies, many ofwhich are suitable for preparing composites or therapy reagentsaccording to the present invention. These include antibodies thatspecifically bind to T-cells, activated T-cells, B-cells, monocytes andgranulocytes, including subpopulations thereof. Certain of theantibodies bind to antigens common to more than one type of leukocyte,e.g., monocytes and granulocytes, B-cells and granulocytes, T-cells andB-cells, T-cells and granulocytes, T-cells and monocytes andgranulocytes and b-cells, and the like. The antibodies produced anddistributed by Immunotech are similar to other antibodies from clonesavailable elsewhere.

Suitable anti-T-cell antibodies include antibodies which bind to theCD1, CD2, CD4, CD6, CD7 or CD8 antigens. Preferred anti-T-cellantibodies are those that bind to the CD3 antigen and the CD5 antigen. Apreferred antibody that binds to both monocyte and granulocyte antigensis a monoclonal which binds in particular to the CDW14 antigen.Preferred antibodies that bind to B-cells include antibodies that bindto the CD19 or CD21 antigens. Antibodies that bind to activated T-cellsinclude monoclonals that bind to the CD25 or CD26 antigens. The CDantigens are leukocyte determinants that define antibodies havingparticular leukocyte specificities. A pair of antibodies that bid to thesame epitope on the same CD antigen will cross-block binding to the sameleukocyte cell types. Antibodies that bind specifically to the Ia(HLA-DR) histocompatibility antigen common to monocyte, B-lymphocytesand activated T-lymphocytes are classified as anti-HLA-DR Class IIantibodies, and are of particular utility for certain applications.

The commercially available monoclonal antibodies to leukocyte antigensare typically of murine or rat origin and typically are IgGs or IgMs,although suitable antibodies for use in preparing conjugates accordingto the invention are not intended to be limited as regards species or Igclass. In general, antibodies can usually be raised to most antigens,using the many conventional techniques now well known in the art. Anyantibody that binds to a leukocyte antigen which is found in sufficientconcentration at a site of inflammation of infection in the body of amammal can be used to make the targeting polyspecific antibody compositefor use in the present invention.

It is generally desirable to use antibodies having a relatively highimmunoreactivity, i.e., a binding constant of at least about 10⁵ 1/mole,preferably at least about 10⁷ 1/mole, and high immunospecificity, i.e.at least about 40%, preferably at least about 60%, more preferably atleast about 70-95% for leukocyte antigens.

It may be preferably for certain applications to use antibodies having asomewhat lower binding constant in the present invention. Antibodieswith high binding constants are likely to bind tightly not only toleukocytes at the side of inflammation or infection but also toleukocytes present in the circulatory system, the marrow or normaltissues. On the other hand, antibodies with a lower binding constantwill tend to accrete mainly at concentrated leukocyte foci at the siteof a lesion, by virtue of a type of mass action effect. This will reducepremature clearance and nontarget accretion of the imaging label or, intherapy applications to be described below, the therapeutic agent, andthus increase the effective amount for targeting the lesion.

Antibody composites for imaging can be prepared by a variety ofconventional procedures, ranging from simply glutaraldehyde linkage tomore elegant and specific linkages between functional groups. Theantibodies and/or antibody fragments are preferably covalently bound toone another, directly or through a short or long linker moeity, throughone or more functional groups on the antibody/fragment, e.g., amine,carboxyl, phenyl, thiol or hydroxyl groups. Various conventional linkersin addition to glutaraldehyde can be used, e.g., disiocyanates,diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimidehydroxysuccinimide esters and the like.

A simple, method is to mix the antibodies/fragments in the presence ofglutaraldehyde to form the antibody composite. The initial Schiff baselinkages can be stabilized, e.g., by borohydride reduction to secondaryamines. This method is conventionally used to prepare other conjugatesof proteins, e.g., peroxidase-antibody conjugates forimmunohistochemical uses or for immunoassays. A diisothiocyanate or acarbodiimide can be used in place of glutaraldehyde as anon-site-specific linker.

Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab')₂ fragments, fusions of more than one clone to formpolyomas that produce immunoglobulins having more than one specificity,and by genetic engineering. The bispecific antibodies can bind to one ormore leukocyte cell types. Bispecific ("hybrid") antibody fragments havebeen prepared by oxidative linkage of Fab' fragments resulting fromreductive cleavage of different antibodies. A portion of these willcontain fragments specific to both of the antigens to which the originalantibodies were raised. This is advantageously carried out by mixing twodifferent F(ab')₂ fragments produced by pepsin digestion of twodifferent antibodies, reductive cleavage to form a mixture of Fab'fragments, followed by oxidative reformation of the disulfide linkagesto produce a mixture of F(ab')₂ fragments including hybrid fragmentscontaining a Fab' portion specific to each of the original antigens.Methods of preparing such hybrid antibody fragments are disclosed inFeteanu, "Labeled Antibodies in Biology and Medicine" pages 321-323(McGraw-Hill Int. Bk. Co, New York et al, 1978); Nisonoff et al, ArchBiochem. Biophys., 93, 470 (1961); and Hammerling et al, J. Exp. Med.,128, 1461 (1968); and in U.S. Pat. No. 4,331,647.

More selective linkage can be achieved by using a heterobifunctionallinker such as a maleimide-hydroxysuccinimide ester. Reaction of thelatter with an antibody/fragment will derivatize amine groups on theantibody/fragment, and the derivative can then be reacted with, e.g., anantibody Fab fragment with free sulfhydryl groups (or a larger fragmentor intact immunoglobulin and sulfhydryl groups appended thereto by,e.g., Traut's Reagent). Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies/fragments at sites remote fromthe antigen binding sites. This can be accomplished by, e.g., linkage tocleaved interchain sulhydryl groups, as noted above. Another methodinvolves reacting an antibody whose carbohydrate portion has beenoxidized with another antibody which has at least one free aminefunction. This results in an initial Schiff base (imine) linkage, whichis preferably stabilized by reduction to a secondary amine, e.g., byborohydride reduction, to form the final composite. Such site-specificlinkages are disclosed, for small molecules or polypeptides or for solidphase polymer supports, in U.S. Pat. No. 4,671,958, and for largeraddends in U.S. Pat. No. 4,699,784.

Included among the various types of bispecific antibody composites ofthe present invention are the following, which are particularly usefulfor certain applications: a composite of antibodies/fragments specificto monocytes and granulocytes for the detection and treatment of, e.g.,osteomyelitis; a composite of antibodies/fragments specific to B-cellsand monocytes for the detection and treatment of, e.g., Crohn's disease;a composite of antibodies/fragments specific to T-cells and B-cells forthe detection and treatment of, e.g., sarcoidosis; a composite ofantibodies/fragments specific to monocytes and lymphocytes for thedetection and treatment of, e.g., tubercular lesions; and a composite ofantibodies/fragments specific to the Ia(DR) histocompatibility antigenand granulocytes for the detection and treatment of infectionsassociated with fever of unknown origin, e.g., granulomatous infections,tubercular lesions, fungal infections and the like.

Similar reactions can be used to bind a plurality of antibodies and/orantibody fragments, e.g., Fab or F(ab')₂ fragments, to one another toform polyspecific composites or composites with more than one epitopicspecificity for a leukocyte cell type to increase its binding affinityor efficiency to the target lesion. Bispecific composites can be linkedto an antibody/fragment specific to a third, fourth or further leukocytecell type using, e.g., a heterobifunctional maleimide-hydroxysuccinimideester linker to derivatize an amine group, followed by reaction of thederivative with a fragment having a free sulfhydryl group, optionallyintroduced with a reagent such as 2-iminothiolane. Alternative linkagemodes will be readily apparent to the ordinary skilled artisan based onthe disclosures for bispecific composite formation, and will requireonly minor variation and adaptation of such methods.

Included among the various types of trispecific or polyspecific antibodycomposites of the present invention are the following, which areparticularly useful for certain applications: a composite ofantibodies/fragments specific to T-cells, B-cells and monocytes for thedetection and treatment of, e.g., graft rejection infiltrates; acomposite of antibodies/fragments specific to B-cells, T-cells,monocytes and granulocytes for the detection and treatment of, e.g.,chronic infection; a composite of antibodies/fragments specific to theIa(DR) antigen, granulocytes and the T-1 antigen for the treatment of,e.g., thyroiditis, graft rejection infiltrates and tubercular lesions.

The antibody composite can be labeled with, or conjugated or adapted forconjugation to, a radioisotope for scintigraphic imaging or a magneticresonance image enhancing agent, for use as a diagnostic imaging agent.Any conventional method of radiolabeling which is suitable for labelingproteins for in vivo use will be generally suitable for labeling thecomposite. This can be achieved by direct labeling with, e.g., aradioisotope of a halogen or a metal ion, using conventional techniquesor more sophisticated methodologies, or by attaching a chelator for aradiometal or paramagnetic ion. Such chelators and their modes ofattachment to antibodies are well known to the ordinary skilled artisanand are disclosed inter alia in, e.g., Childs et al., J. Nuc. Med.,26:293 (1985); and in Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376,4,361,544, 4,468,457, 4,444,744, and 4,624,846. Typical are derivativesof ethylenediaminetetraacetic acid (EDTA) anddiethylenetriaminepentaacetic acid (DPTA). These typically have groupson the side chain by which the chelator can be attached to an antibody.Alternatively, carboxyl or amine groups on a chelator can be activatedand then coupled to an antibody composite by well known methods. Forexample, deferoxamine, which is a chelator for Ga-67 has a free aminegroup that can be activated with a suitable linker to contain anactivated carboxyl, isothiocyanate or like group, and then coupled toamines on an antibody composite.

The chelator may be bound to the antibody composite, directly or througha short or long chain linker moiety, through one or more functionalgroups on the antibody, e.g., amine, carboxyl, phenyl, thio or hydroxylgroups. Various conventional linkers can be used, e.g., diisocyanates,diisothiocyanates, carbodiimides, bis-hydroxysuccinimide esters,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like,preferably a selective sequential linker such as theanhydride-isothiocyanate linker disclosed in U.S. Pat. No. 4,680,338.

Labeling with either Iodine-131 (I-131) or Iodine-123 (I-123) is readilyeffected using an oxidative procedure wherein a mixture of radioactivepotassium or sodium iodide and the antibody is treated withchloramine-T, e.g., as reported by Greenwood et al, Biochem. J., 89, 114(1963) and modified by McConahey et al, Int. Arch. Allergy Appl.Immunol., 29, 185 (1969). This results in direct substitution of iodineatoms for hydrogen atoms on the antibody molecule, presumable ontyrosine residues, possibly also on tryptophan and even on phenylalanineresidues, depending on the proportions of reagents and the reactionconditions. Alternatively, lactoperoxidase iodination may be used, asdescribed by Feteanu, supra, page 303, and references cited therein.

Some more advanced methods of labeling are disclosed in pendingapplications U.S. Ser. Nos. 742,436 (6-7-85), 084,544 (8-12-87), and176,421 (4-1-88). The disclosures of all of the foregoing patents andapplications are incorporated herein in their entireties by reference. Awide range of labeling techniques are disclosed in Feteanu, "LabeledAntibodies in Biology and Medicine", pages 214-309 (McGraw-Hill Int.Book Co., New York et al, 1978). The introduction of various metalradioisotopes may be accomplished according to the procedures of Wagneret al., J. Nucl. Med., 20,428 (1979); Sundberg et al, J. Med. Chem., 17,1304 (1974); and Saha et al. J. Nucl. Med., 6, 542 (1976). The foregoingare merely illustrative of the many methods of radiolabeling proteinsknown to the art.

Examples of compounds useful for MRI image enhancement includeparamagnetic ions, e.g., Gd(III), Eu(III), Dy(III), Pr(III), Pa(IV),Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III) and V(IV)ions, or radicals, e.g., nitroxides, and these would be conjugated to asubstrate bearing paramagnetic ion chelators or exposed chelatingfunctional groups, e.g., .SH, NH₂, COOH, for the ions, or linkers forthe radical addends. The MRI enhancing agent must be present insufficient amounts to enable detection by an external camera, usingmagnetic field strengths, which are reasonably attainable and compatiblewith patient safety and instrumental design. The requirements for suchagents are well known in the art for those agents which have effect uponwater molecules in the medium, and are disclosed, inter alia, in, e.g.,Pykett, Scientific American, 246:78 (1982); and Runge et al., Am. J.Radiol., 141:1209 (1987).

It is well understood that many of the same methods for introducingmetals, directly or in the form of chelates, into antibodies will besuitable for introduction of MRI agents into the antibody composites ofthe invention to form imaging agents for infectious lesions. MRI agentsadvantageously have a large number of paramagnetic ions or radicals forenhanced imaging. One method for introducing a pluralityof such ions isto load a carrier polymer with chelates and link the carrier to theantibody composite, preferably site-specifically at a site remote fromthe antigen binding sites of the composite. This has the advantage thatlarger numbers of chelators can be attached to the antibody at fewersites on the antibody itself, so that immunoreactivity is not asseriously compromised. Examples of polymers that are useful for loadingthe antibody with chelator include, e.g., polyols, polysaccharides,polypeptides and the like. See U.S. Pat. Nos. 4,699,784 (Shih et al) and4,046,722 (Rowland). One type of polysaccharide is dextran. The chelatorcan be functionalized to contain reactive groups towards the dextranhydroxyls, e.g., anhydrides, isocyanates or isothiocyanates and thelike. Alternatively, dextran can be derivatized in a number of ways,e.g., by conversion to an aminodextran. It will be appreciated thatsimilar methods will be useful for loading a plurality of drug moleculeson an antibody or antibody composite, as will be discussed more fullyhereinafter.

The process for preparing an antibody conjugate with an aminodextran(AD) carrier normally starts with a dextran polymer, advantageously adextran of average molecular weight (MW) of about 10,000-100,000,preferably about 10,000-40,000, and more preferably about 15,000. Thedextran is then reacted with an oxidizing agent to effect a controlledoxidation of a portion of its carbohydrate rings to generate aldehydegroups. The oxidation is conveniently effected with glycolytic chemicalreagents, e.g., NaIO₄, according to conventional procedures.

It is convenient to adjust the amount of oxidizing agent so that about50-150, preferably 100 aldehyde groups are generated, for a dextran ofMW of about 40,000, with about the same proportion of aldehyde groupsfor other MW dextrans. A larger number of aldehyde groups, andsubsequent amine groups, is less advantageous because the polymer thenbehaves more like polylysine. A lower number results in less desirableloading of the chelator or boron addend, which may be disadvantageous.

The oxidized dextran is then reacted with a polyamine, preferably adiamine, and more preferably a mono- or poly-hydroxy diamine. Suitableamines include, e.g., ethylenediamine, propylenediamine or similarpolymethylenediamines, diethylenetriamine or like polyamines,1,3-diamino-2-hydroxypropane or other like hydroxylated diamines orpolyamines, and the like. An excess of the amine relative to thealdehyde groups can be used, to insure substantially complete conversionof the aldehyde functions to Schiff base (imine) groups.

Reductive stabilization of the resultant intermediate is effected byreacting the Sciff base intermediate with a reducing agent, e.g., NaBH₄,NaBH₃ CN, or the like. An excess of the reducing agent is used to assuresubstantially complete reduction of the imine groups to secondary aminegroups, and reduction of any unreacted aldehyde groups to hydroxylgroups. The resultant adduct can be further purified by passage througha conventional sizing column to remove cross-linked dextrans. Anestimate of the primary number of available amino groups on the AD canbe effected by reaction of a weighed sample with trinitrobenzenesulfonicacid and correlation of the optical density at 420 nm with a standard.This method normally results in essentially complete conversion of thecalculated number of aldehyde groups to primary amine groups on the AD.

Alternatively, the dextran can be derivatized by conventional methodsfor introducing amine functions, e.g., by reaction with cyanogenbromide, followed by reaction with a diamine. The AD should be reactedwith a derivative of the particular drug or chelator, in an activatedform, preferably a carboxyl-activated derivative, prepared byconventional means, e.g., using dicyclohexylcarbodiimide (DCC) or awater soluble variant thereof.

The scintigraphic imaging method of the invention is practiced byinjecting a mammal, preferably a human, parenterally with an effectiveamount for scintigraphic imaging of the radiolabeled polyspecificanti-leukocyte antibody composite. By parenterally is meant, e.g.intravenously, intraarterially, intrathecally, interstitially orintracavitarily. It is contemplated that a subject will receive a dosageof from about 1 mCi to 50 mCi of radiolabeled conjugate, the amountbeing a function of the particular radioisotope and mode ofadministration. For intravenous injection, the amounts are normally:about 2-10 mCi, preferably about 2-5 mCi, of I -131; about 5-10 mCi,preferably about 8 mCi, of I-123; about 10-40 mCi, preferably about 20mCi of Tc-99 m; about 2-5 mCi, preferably about 4 mCi of in-111 orGa-67.

The radiolabeled polyspecific anti-leukocyte antibody composite isconveniently provided as an injectable preparation for mammalian use,preferably a sterile injectable preparation for human use, for targetinga scintigraphic imaging agent to an infectious or inflammatory lesioncontaining leukocytes, preferably comprising: a sterile injectablesolution containing an effective amount of the radiolabeled composite ina pharmaceutically acceptible sterile injection vehicle, preferablyphosphate-buffered saline (PBS) at physiological pH and concentration.Other conventional pharmaceutically acceptable vehicles may be utilizedas required for the site of parenteral administration.

A representative preparation to be parenterally administered inaccordance with this invention will normally contain about 0.1 to 20 mg,preferably about 2 mg, of radiolabeled polyspecific antibody composite,in a sterile solution which advantageously also contains, e.g., about 10mg of human serum albumin (1% USP: Parke-Davis) per milliliter of 0.04Mphosphate buffer (pH 7.4 Bioware) containing 0.9% sodium chloride.

Once enough isotope has deposited at the target site, scanning iseffected with either a conventional planar and/or SPECT gamma camera, orby use of a hand held gamma probe used externally or internally tolocalize the inflammation or the lesion. The scintigram is normallytaken by a gamma imaging camera having one or more windows for detectionof energies in the 50-500 KeV range. The target site can be anyinfectious lesion, inflammatory deposit or occult lesion havingleukocytes present in a relatively concentrated focus. Localization oflesions containing leukocytes will occur directly through reactivity ofthe polyspecific antibody-agent conjugate with the leukocytes residentin the lesion at the time of parenteral administration as well asthrough entry of labeled leukocytes into the lesion.

Magnetic resonance imaging (MRI) is effected in an analogous method toscintigraphic imaging except that the imaging agents will contain MRIenhancing species rather than radioisotopes. It will be appreciated thatthe magnetic resonance phenomenon operates on a different principle fromscintigraphy. Normally the signal generated is correlated with therelaxation times of the magnetic moments of protons in the nuclei of thehydrogen atoms of water molecules in the region to be imaged. Themagnetic resonance image enhancing agent acts by increasing the rate ofrelaxation, thereby increasing the contrast between water molecules inthe region where the imaging agent accretes and water moleculeselsewhere in the body. However, the effect of the agent is to increaseboth T₁, and T₂, the former resulting in greater contrast, while thelatter results in lesser contrast. Accordingly the phenomenon isconcentration-dependent, and there is normally an optimum concentrationof a paramagnetic species for maximum efficacy. The optimumconcentration will vary with the particular agent used, the locus ofimaging, the mode of imaging, i.e., spin-echo, saturation-recovery,inversion-recovery and for various other strongly T₁ dependent or T₂dependent imaging techniques, and the composition of the medium in whichthe agent is dissolved or suspended. These factors, and their relativeimportance are known in the art. See, e.g., Pykett, op.cit., and Rungeet al., op.cit.

The MRI method of the invention is practiced by injecting a mammal,preferably a human, parenterally with an effective amount for magneticresonance imaging of a conjugate according to the present invention of apolyspecific anti-leukocyte antibody composite and an MRI enhancingagent. It is contemplated that a subject will receive a dosage oflabeled conjugate sufficient to enhance the MRI signal at the site of alesion by at least about 20%, preferably 50-500%, the amount being afunction of the particular paramagnetic species and the mode ofadministration.

Again, the labeled polyspecific anti-leukocyte antibody composite isconveniently provided as an injectable preparation for mammalian use,preferably a sterile injectable preparation for human use, for targetinga MRI agent to an infectious or inflammatory lesion containingleukocytes, preferably comprising: a sterile injectable solutioncontaining an effective amount of the labeled composite in apharmaceutically acceptable sterile injection vehicle, preferablyphosphate-buffered saline (PBS) at physiological pH and concentration.Other conventional pharmaceutically acceptable vehicles for parenteraladministration may be utilized as required for the site of parenteraladministration.

A representative preparation to be parenterally administered inaccordance with this invention will normally contain about 0.1 to 20 mg,preferably about 2 mg, of labeled polyspecific antibody composite, in asterile solution which advantageously also contains, e.g., about 10 mgof human serum albumin (1% USP: Parke-Davis) permilliliter of 0.04Mphosphate buffer (pH 7.4 Bioware) containing 0.9% sodium chloride. Onceenough of the MRI agent has deposited at the target site, scanning iseffected with a conventional MRI camera camera to image the lesion.

In a preferred embodiment of this invention, the localization ratio ofthe primary labeled polyspecific antibody-agent conjugate is enhancedthrough the use of a nonlabeled second antibody to scavenge non-targetedcirculating conjugate and promote its clearance, as disclosed forrelated imaging agents in Goldenberg, U.S. Pat. No. 4,624,846, thedisclosure of which is incorporated herein in its entirety by reference.This technique is likewise applicable to the polyspecific anti-leukocyteantibody composite conjugated to a thereapeutic drug, as will bediscussed hereinafter. The term "localization ratio" is utilized in itsconventional sense, i.e. The ratio of target to non-target antibodyconjugate. In general, the second antibody is used in an amount thatwill enhance the localization ratio of the primary antibody conjugate byat least about 20 percent and typically by 50 percent or more.

The second antibody may be whole IgG or IgM, or a fragment of IgG orIgM, so long as it is capable of binding the primary antibody conjugateto form a complex which is cleared from the circulation and thenon-target spaces more rapidly than the primary antibody conjugate byitself. Preferably, the second antibody will be whole IgG or IgM. If theprimary antibody is a fragment of IgG or IgM, it is preferable that thesecond antibody be whole IgG or IgM so that the primary/secondarycomplex retains the capability of activating the complement cascade.Conversely, where the primary antibody is whole IgG, the second antibodymay be a fragment if the complex still retains complement-fixingcapability. It is preferred that at least one of the primary/secondarypair be whole IgG or IgM. one advantage of using IgM is that it forms ahigher molecular weight complex with primary antibody or with detachedconjugates, i.e., diagnostic and/or therapeutic principles such asdrugs, chelating agents, radionuclides, and the like. This will increasethe rate and effectiveness of clearance of non-target primary antibodyand/or principle, especially from blood. The second antibody can beprepared by methods disclosed in the aforementioned Goldenberg '846patent. Monoclonal anti-species IgG is also available and isadvantageously used as second antibody in the present process.Non-metallic conjugates, e.g., radioiodinated linking groups or organicparamagnetic species such as nitroxides, can also be haptens to whichthe second antibody is specific.

The second antibody is injected into the subject after a sufficient timehas elapsed following parenteral administration of the primarypolyspecific antibody-agent conjugate to permit maximum uptake thereofby leukocytes, typically about 2-72 hours following the initialadministration preferably at about 24-48 hours post-administration. Ifthe primary antibody is not administered intravenously, it may beadvantageous to administer at least a portion of the second antibody bythe same parenteral route. It is advantageous however, to inject atleast a portion of the second antibody intraveneously to accelerateclearance of primary antibody which has diffused into the circulatorysystem.

The use of second antibody to clear circulating labeled primary antibodyand enhance the localization ratio of the primary antibody is furtherenhanced by utilization of image-enhancing subtraction techniques asdisclosed in the foregoing Goldenberg patents as well as the referencescited therein. This is an art-recognized technique wherein anindifferent antibody or fragment labeled with a radionuclide capable ofindependent detection. This antibody has substantially the same kineticsof distribution and metabolism as the primary antibody during the periodrequired for imaging. The injection of such antibodies is preferred overconventional subtraction agents, such as Tc-99m-labeled serum albumin,which are nevertheless suitable for use to enhance image processing bycompensating for background. The use of the radiolabeled indifferentantibody as a subtraction agent permits computerized correction fornontarget background radiation from organs which effect clearance ofantibodies from the circulatory system. It will be appreciated by thoseof ordinary skill in the art that the primary monoclonal antibody andthe indifferent antibody utilized as a subtraction agent are preferablyfrom the same species or myeloma/hybridoma so that the second antibodywill clear the primary monoclonal antibody and the indifferent antibodyimmunoglobulin from untargeted areas at substantially the same rate. Itis further preferred that the second antibody be specific to a constantregion of the primary and indifferent immunoglobulin species.

The amount of second antibody introduced will generally be that amountwhich can decrease the circulating primary antibody by 10-85% within2-72 hours. The ratio of second antibody to primary antibody which willaffect the clearance will depend upon the binding properties of theprimary and secondary antibody pair. Preliminary screening of patientblood in vitro can be used to provide an initial estimate of theappropriate ratio. The screen will be used to determine the ratio ofsecond antibody to primary antibody required to obtain a precipitin bandin, e.g., a gel diffusion test. This indicates the general range of themolar ratio of second antibody to primary antibody, which serves as ameasure of the lower limit for the ratio, since in vivo application mayrequire a higher ratio of second antibody to primary antibody than isindicated by such in vitro tests.

In practice, the molar ratio of second antibody to primary antibody willgenerally be in the range of about 5-50, although the range should notbe considered limitative. Molar ratios of second antibody to primaryantibody of 15-25, and preferably 20-25, have been found to beadvantageous where both the primary and the second antibody are wholeIgG.

Many drugs are known which have a cytotoxic effect on cells ormicroorganisms that may infect a human and cause a lesion. They can befound in any of the readily available art-recognized compendia of drugsand toxins, such as the Merck Index and the like. Any such antibioticdrug can be conjugated to an anti-leukocyte antibody or antibodycomposite to form a therapy agent according to the present invention,and the use of such a conjugate to improve the targeting of an antiboticdrug to the site of an infectious lesion so as to increase its effectiveconcentration of the site is a part of the present invention. One ormore antibiotic drugs is/are conjugated to a polymeric carrier which isthen conjugated to the antibody or antibody composite, for therapeuticuse. In certain cases, it is possible to partially or completelydetoxify a drug as part of the antibody conjugate, while it is incirculation, which can reduce systemic side effects of the drug andpermit its use when systemic administration of the drug would beunacceptable. Administration of more molecules of the drug conjugated toa polymer which is further conjugated to the antibody, permits therapywhile mitigating systemic toxicity.

The methodology of this invention is applicable to the therapeutictreatment of infectious lesions by conjugating the primary antibody orantibody composite to an antibiotic drug. Art recognized methods ofconjugating antibiotic drugs to immunoglogulins are described, e.g., in:the chapter by O'Neill, entitled "The Use of Antibodies as DrugCarriers," in Drug Carriers in Biology and Medicine, G. Gregoriadis,ed., Academic Press London, 1979; Arnon et al., Recent Results in CancerRes. 75:236 1980; and Moolton et al., Immunolog. Res. 62:47, 1982,showing art awareness. These methods are quite similar to the methods,employed for coupling drugs effective against various disease-causingmicroorganisms, such as against bacteria, viruses, fungi and diverseparasites to antibodies developed against these microorganisms, theirproducts or antigens associated with their lesions.

Such antibaterial, antiviral, antiparasitic, antifungal and relateddrugs, e.g., sulfonamides, penicillins and cephalosporins,animoglycosides, tetracyclines, chloramphenicol, piperazine,chloroquine, diaminopyridines, metroniazide, isoniazide, rifampins,streptomycins, sulfones, erythromycin, polymixins, nystastin,amphotericins, 5-fluorocytosine, 5-iodo-2'-deoxyuridine,1-adamantanamine, adenine arabinoside, ananitins and azidothymidine(AZT), are preferred for coupling to appropriate specificantibodies/fragments and antibody/fragment composites. Various otherpotential antimicrobial agents for use in this invention are listed inGoodman et al., "The Pharmacological Basis of Therapeutics," SixthEdition, A. G. Gilman et al., eds., Macmillan Publishing Co., New York,1098, showing general art awareness. Various conditions appropriate anddesirable for targeting drugs to specific target sites have beenreviewed e.g. by Trouet et al., in Targeting of Drugs, G. Gregoriadis etal., eds., Plenum Press, New York and London, 1982, pp. 19-30, showingclinical knowledge of how such targeting would benefit patientssuffering from infectious lesions.

The use of a second antibody as described above will increase theeffectiveness of the therapeutic agent according to the invention in thesame manner as for the diagnostic imaging conjugate. The effectivenessof the therapeutic agent is expressed in terms of its therapeutic indexwhich, utilized in the conventional sense, is defined as the ratio oftherapeutic effects to undesirable side effects. It is often defined interms of a quantitative measure of efficacy vs. toxicity in a standardmodel system, e.g., the ratio of the median lethal dose (LD₅₀) to themedian effective dose (ED₅₀). The use of second antibody as describedherein produces an increase in the therapeutic index of anti-leukocyteantibody and antibody composite conjugates by clearing nontarget primaryantibody and/or detached therapeutic principle. In addition to beingspecific to the primary monoclonal antibody as discussed above, in theinstance of the therapeutic preparation, the second antibody can bespecific to the therapeutic agent. It can also be specific to a carrierfor the therapeutic agent.

Therapeutic preparations contemplated herein comprise monospecificanti-leukocyte antibodies/fragments as defined above, conjugated to atherapeutically effective drug, in a suitable vehicle for parenteraladministration. A therapeutic preparation may likewise comprise apolyspecific anti-leukocyte antibody/fragment composite conjugated to anantibiotic. Therapeutic preparations may also include a separatelypackaged second antibody as described above. Suitable vehicles are wellknow in the art and can include, e.g., analogous sterile PBS solutionsto those used for administration of diagnostic imaging agents, asdiscussed hereinabove.

The anti-leukocyte polyspecific imaging conjugates and monospecific orpolyspecific therapeutic conjugates according to the invention also canbe conveniently provided in a therapeutic or diagnostic kit for antibodytargeting to an infectious or inflammatory lesion containing a focus ofleukocytes. Typically, such a kit will comprise: a vial containing theantibody conjugate of the present invention, either as a lyophilizedpreparation or in an injection vehicle; if the conjugate is to be usedfor scintigraphic imaging, it will generally be provided as a coldconjugate together with reagents and accessories for radiolabeling, inseparate containers, while MRI agents and therapeutic conjugates willgenerally be supplied with a paramagnetic species or an antibioticalready conjugated to the antibody/fragment composite or monospecificantibody/fragment. The kit may further contain a second, separatelypackaged, unlabeled antibody or antibody fragment specific against theantibody or fragment or the therapeutic agent, a carrier therefor, or achelating agent for the radionuclide or paramagnetic ion.

The imaging preparations and methods of this invention are at least asefficacious as the conventional agents for determination of occultabscesses using In-111-labeled leukocytes and are clearly advantageousthereover in terms of cost, potential toxicity of the reagent and, mostsignificant, ease of use. The therpeutic reagents and methods of theinvention provide a means to target sites of microbial infection withantibiotic drugs to improve the therapeutic index of the drugs, reducesystemic side effects and enhance their efficacy.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments aretherefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsover. In the followingexamples, all temperatures are set forth uncorrected in degrees Celsius,unless otherwise indicated, all parts and percentages are by weight.

EXAMPLE 1 Bispecific Anti-leukocyte Composite

A bispecific antibody composite is prepared from a F(ab')₂ fragment of amonoclonal antibody highly specific for granulocyte cells. Theinterchain disulfide bridges are reduced carefully with cysteine, takingcare to avoid light-heavy chain cleavage, to form Fab'-SH fragments. TheSH group(s) is(are) activated with an excess of bis-maleimide linker(1,1'-(methylenedi-4,1-phenylene)bis-maleimide, Aldrich Chemical Co.,Milwaukee, WI.). A second monoclonal antibody, highly specific for anantigen common to B-lymphocyte cells and macrophages (anti-Ia), isconverted to Fab'-SH and then reacted with the activatedanti-granulocyte fragment to obtain a bispecific composite. Thecomposite can be reacted with 2-iminothiolane to introduce one or moresulfhydryl groups for use in coupling the composite to a thirdantibody/fragment, using the same bis-maleimide activation proceduredescribed above, or for use in direct metallation with, e.g., Tc-99mfrom reduced (e.g., with SnC1₂) pertechnetate.

EXAMPLE 2 Polyspecific Anti-leukocyte Composite

A polyspecific antibody composite is prepared from a monoclonal antibodyspecific for T-lymphocytes (anti-T-1), which has been converted toFab'-SH fragments and activated with bis-maleimide linker, then coupledto the bispecific fragment described above that has been activated byincorporation of an SH group, using 2-iminothiolane. The polyspecificcomposite is labeled with one of the several radionuclides useful forimaging (Tc-99m, I-123, Ga-67, In-111) for use to detect infectiouslesions or inflammatory deposits by RAID.

EXAMPLE 3 Scintigraphic Imaging Kit

A diagnostic imaging kit contains: a first sterile vial fitted with arubber septum, and containing the thiol-activated polyspecific compositeof Example 2, in the form of a lyophilized preparation; a second sterile(septum-sealed) vial containing phosphate-buffered saline, pH 7.4; athird sterile (septum-sealed) vial containing a solution of stannousglucoheptonate for reduction of 99m-pertechnetate; and additionalseptum-sealed sterile vials and sterile syringes for labeling andinjection of the preparation. Optionally, the kit can include a separatesterile vial containing a second antibody for rapid clearance ofcirculating labeled composite after localization, for example,affinity-purified rabbit anti-mouse antibody.

EXAMPLE 4 Diagnostic Imaging

A 24-year old female patient develops fever and abdominal pain one weekafter giving birth to a male infant by Caesarean section. The patient ismaintained on I.V. antibiotic therapy for two weeks, but the fever andabdominal pain persists. CAT scans fail to demonstrate any abnormalmass. An immunoscintigraphy study is performed using the polyspecificanti-leukocyte composite of Example 2, directly labeled with Tc-99mradioisotope using the kit components of Example 3 andgenerator-produced sodium pertechnetate. An injection of 20 mCi ofradiolabeled composite is used, and the patient is scanned with a gammacamera in SPECT mode. The scan of the patient's abdomen demonstrates afocus of accumulation of Tc-99m. Surgery is performed and an abscess isfound at the site of Tc-99m activity. The abscess is drained, andpathology demonstrates large numbers of granulocytes, as well asmonocytes, B-lymphocytes and activated T-lymphocytes, present in thepurulent material. After two days, the patient's fever and pain subside.

EXAMPLE 5 Diagnostic Imaging

A 58-year old female patient that has been treated for pyelonephritisdevelops fever and acute spinal tenderness. Vertebral osteomyelitis issuspected, but radiography of the spine is normal. An immunoscintigraphystudy is performed using the polyspecific anti-leukocyte composite ofExample 2, directly labeled with Tc-99m radioisotope using the kitcomponents of Example 3 and generator-produced sodium pertechnetate. Aninjection of 20 mCi of radiolabeled composite is used, and the patientis scanned with a gamma camera in planar imaging mode 24 hours afteradministration of the labeled composite. The scan shows an intense focusof Tc-99m just above the point of termination of the spinal chord.Laminectomy and drainage of the epidural spaces is performed at thetarget site. Pathology of the purulent material in the drainage fluiddemonstrates primarily exhuberant granulation tissue containing largenumbers of mononuclear lymphoid cells with fewer numbers ofgranulocytes.

EXAMPLE 6 Therapy

A 42-year old male with AIDS presents with bilateral pneumonia whichfails to respond to conventional broad-spectrum antibiotic therapy. Theclinical presentation and spectrum cytology suggest Pneumocystis cairniipneumonia which appears to be advanced and to be causing the patientsevere respiratory distress. A mixture of monoclonal antibodies whichbind to granulocyte and monocyte antigens are each site-specificallyconjugated to an average of one aminodextran carrier of about 15,000 MW,bearing an average of about 25 molecules of trimethoprin (Wellcome), amethotrexate derivative conjugated to the carrier by adapting themethodology of the Shih et al. patent cited hereinabove. A slowintravenous infusion of about 25 mg of the antibody conjugate mixturedelivers a therapeutic dose of the antibiotic to the pulmonary lesions,and is repeated on each of three sucessive days. Two days later, thepatient begins to show improvement and his fever abates, and this isconfirmed by improvement in his chest roentgenograms three days later.Most of the pneumonia resolves within two weeks of the antibody-drugconjugate treatment, with the patient receiving general supportivemeasures during this time.

The preceding examples can be repeated with similar success bysubstituting the generically or specificially described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A polyspecific anti-leukocyte antibody conjugatefor targeting foci of leukocyte accretion, comprising an immunoreactivepolyspecific composite of at least two different substantiallymonospecific antibodies or antibody fragments, conjugated to at leastone imaging agent, wherein at least two of said antibodies or antibodyfragments specifically bind to different leukocyte cell types.
 2. Theanti-leukocyte conjugate of claim 1, wherein said at least two leukocytecell types are at least two of granulocytes, monocytes, B-lymphocytesand T-lymphocytes.
 3. The anti-leukocyte conjugate of claim 1, whereineach said antibody or antibody fragment is a monoclonal antibody orfragment.
 4. The anti-leukocyte of claim 1, wherein said substantiallymonospecific antibody fragments are F(ab')₂, Fab' or Fab fragments. 5.The anti-leukocyte conjugate of claim 4, wherein said antibody fragmentsare each fragments of monoclonal antibodies.
 6. The anti-leukocyteconjugate of claim 1, wherein said imaging agent is a radioisotopeemitting gamma in the range of 50-500 KeV.
 7. The anti-leukocyteconjugate of claim 6, wherein said radioisotope is Tc-99m, I-123, Ga-67or In-111.
 8. The anti-leukocyte conjugate of claim 1, wherein saidimaging agent is a magnetic resonance image enhancing agent.
 9. Theanti-leukocyte conjugate of claim 1, wherein at least one of saidantibodies or antibody fragments specifically binds to the Ia(DR)antigen.
 10. A method for targeting an imaging agent to an inflammatoryor infectious lesion, which comprises injecting a mammal parenterallywith an effective amount for targeting of the anti-leukocyte conjugateof claim
 1. 11. The method of claim 10, wherein said imaging agent is aradioisotope, and wherein said method further comprises obtaining ascintigraphic image of said lesion after a time sufficient for saidanti-leukocyte conjugate to localize at the site of said lesion.
 12. Themethod of claim 10, wherein said imaging agent is a magnetic resonanceimage enhancing agent and wherein said method further comprisesobtaining a magnetic resonance image of said lesion after a timesufficient for said anti-leukocyte conjugate to localize at the site ofsaid lesion.
 13. The method of claim 10, additionally comprising thestep of parenterally administering to the subject, at a time afteradministration of said anti-leukocyte conjugate sufficient to permitmaximum selective uptake thereof by leukocytes at the site of saidlesion, a second, unlabeled antibody or antibody fragment whichspecifically binds to said anti-leukocyte conjugate, in an amountsufficient to increase the localization ratio of said anti-leukocyteconjugate by at least about 20 percent within a period of from 2 to 72hours.
 14. A sterile injectable preparation for human use, for targetingan imaging agent to an infectious or inflammatory lesion containingleukocytes, comprising an effective amount for imaging of thepolyspecific anti-leukocyte conjugate of claim 1, in a pharmaceuticallyacceptible sterile injection vehicle.
 15. A kit suitable for use in thein vivo detection of an infectious or inflammatory lesion containingleukocytes, comprising, in a suitable container, the anti-leukocyteconjugate of claim
 1. 16. The kit of claim 15, additionally comprising,in a second container, a second, unlabeled antibody or antibody fragmentwhich specifically binds to said anti-leukocyte conjugate.